Paolo Orsini

Two-photon polymerization of sub-micrometric patterned surfaces: investigation of cell-substrate interactions and improved differentiation of neuron-like cells

Direct Laser Writing (DLW) is an innovative tool that allows the photofabrication of high resolution 3D structures, which can be successfully exploited for the study of the physical interactions between cells and substrates. In this work, we focused our attention on the topographical effects of submicrometric patterned surfaces fabricated via DLW on neuronal cell behavior. In particular, we designed, prepared, and characterized substrates based on aligned ridges for the promotion of axonal outgrowth and guidance. We demonstrated that both rat PC12 neuron-like cells and human SH-SY5Y derived neurons differentiate on parallel 2.5 μm spaced submicrometric ridges, being characterized by strongly aligned and significantly longer neurites with respect to those differentiated on flat control substrates, or on more spaced (5 and 10 μm) ridges. Furthermore, we detected an increased molecular differentiation toward neurons of the SH-SY5Y cells when grown on the submicrometric patterned substrates. Finally, we observed that the axons can exert forces able of bending the ridges, and we indirectly estimated the order of magnitude of these forces thanks to scanning probe techniques. Collectively, we showed as submicrometric structures fabricated by DLW can be used as a useful tool for the study of the axon mechanobiology.

Weak hydrostatic forces in far-scanning ion conductance microscopy used to guide neuronal growth cones

Scanning ion conductance microscopy (SICM) is currently used for high resolution topographic imaging of living cells. Recently, it has been also employed as a tool to deliver stimuli to the cells. In this work we have investigated the mechanical interaction occurring between the pipette tip and the sample during SICM operation. For the purpose, we have built a setup combining SICM with atomic force microscopy (AFM), where the AFM cantilever replaces the sample. Our data indicate that, operating in far-scanning mode with current decrease values below 2%, no force can be detected, provided that the level of the electrolyte filling the pipette is equal to that determined by the capillary tension. A filling level different from this value determines a hydrostatic pressure, a flux through the pipette tip and detectable forces, even in far-scanning mode. The absolute value of these forces depends on the pipette tip size. Therefore, a possible pitfall when using SICM for cell imaging is to imply zero-force working conditions. However the hydrostatic forces can be exploited in order to deliver weak mechanical stimuli and guide neuronal growth cones. Evidences of the effectiveness of this approach are herein given.

Use of scanning ion conductance microscopy to guide and redirect neuronal growth cones

Scanning ion conductance microscopy has been applied to neuronal growth cones of the leech either to image or to stimulate them. Growth cone advance was recorded in non-contact mode using a 2% ion current decrease criterion for pipette-membrane distance control. We demonstrate effective growth cone remodelling using a 5% criterion (near-scanning). Recurrent line near-scanning aligned growth cone processes along the scan line. The new membrane protrusions, marked by DiI, started a few minutes after scanning onset and progressively grew in thickness. Using scanning patterns suitable for connecting distinct growth cones, new links were consistently developed. Although the underlying mechanism is still a matter for investigation, a mechanical perturbation produced by the moving probe appeared to induce the process formation. Thanks to its deterministic and interactive features, this novel approach to guiding growth cones is a promising way to develop networks of identified neurons as well as link them with artificial structures.

Effects of vestibular and neck proprioceptive stimulation on posture as a function of hypnotizability.

Abstract Previous studies on the role of hypnotizability in postural control indicate that the body sway of subjects with high or low hypnotizability to hypnosis is differentially modulated by eye closure. The aim of this study was to investigate whether hypnotizability also modulates the postural response to electrical vestibular stimulation and to head rotation in nonhypnotized individuals. The center of pressure (CoP) displacements were monitored in highs and lows standing on a stabilometric platform with closed eyes during basal conditions and electrical vestibular stimulation in 3 different positions of the head. Results showed that the CoP stimulus-locked displacements as well as the CoP mean position, area, and mean velocity were similar in highs and lows, but only in lows did the head position modulate the mean velocity. This finding might reflect a difference in sensory-motor integration between the 2 groups.

Can imagery become reality

Previous studies showed that highly hypnotizable persons imagining a specific sensory context behave according to the corresponding real stimulation and perceive their behaviour as involuntary. The aim of the study was to confirm the hypothesis of a translation of sensory imagery into real perception and, thus, of a true involuntary response. We studied the imagery-induced modulation of the vestibulospinal (VS) reflex earlier component in highly (Highs) and low hypnotizable subjects (Lows), as it is not affected by voluntary control, its amplitude depends on the stimulus intensity, and the plane of body sway depends on the position of the head with respect to the trunk. Results showed that the effects of the “obstructive” imagery of anaesthesia are different from those elicited by the “constructive” imagery of head rotation. Indeed, both Highs and Lows having their face forward and reporting high vividness of imagery experienced anaesthesia and reduced their VS reflex amplitude in the frontal plane, while only Highs changed the plane of body sway according to the imagined head rotation that is from the frontal to the sagittal one. These effects cannot be voluntary and should be attributed to translation of sensory imagery into the corresponding real perception.

Measuring the elastic properties of living cells through the analysis of current–displacement curves in scanning ion conductance microscopy

Knowledge of mechanical properties of living cells is essential to understand their physiological and pathological conditions. To measure local cellular elasticity, scanning probe techniques have been increasingly employed. In particular, non-contact scanning ion conductance microscopy (SICM) has been used for this purpose; thanks to the application of a hydrostatic pressure via the SICM pipette. However, the measurement of sample deformations induced by weak pressures at a short distance has not yet been carried out. A direct quantification of the applied pressure has not been also achieved up to now. These two issues are highly relevant, especially when one addresses the investigation of thin cell regions. In this paper, we present an approach to solve these problems based on the use of a setup integrating SICM, atomic force microscopy, and optical microscopy. In particular, we describe how we can directly image the pipette aperture in situ. Additionally, we can measure the force induced by a constant hydrostatic pressure applied via the pipette over the entire probe–sample distance range from a remote point to contact. Then, we demonstrate that the sample deformation induced by an external pressure applied to the pipette can be indirectly and reliably evaluated from the analysis of the current–displacement curves. This method allows us to measure the linear relationship between indentation and applied pressure on uniformly deformable elastomers of known Young’s modulus. Finally, we apply the method to murine fibroblasts and we show that it is sensitive to local and temporally induced variations of the cell surface elasticity.

A prevalence study of known diabetes mellitus in Tuscany assessed from pharmaceutical prescriptions and other independent sources.

This study evaluates the prevalence of diabetes mellitus (DM) in Pisa (Tuscany, Italy) using four independent data sources. The main source, represented by computerized prescriptions for anti-diabetic agents collected over a 4-month period, was validated using three secondary sources: (a) the list of diabetic patients who receive material of self-care from the National Health Service; (b) the clinical records of diabetic patients obtained from a random sample of family doctors; (c) the clinical records of diabetic patients attending our outpatient clinic. The main source provided 3806 patients, and 697 patients were added from the secondary sources, thus identifying a total number of 4503. The prevalence of known DM in the “Pisa area” exclusively reckoned by the main source, was 2.01%, and the prevalence corrected by the addition of the various sources resulted in 2.4%. The capture-recapture method showed a completeness of ascertainment of the survey of 90.1%, and thus an estimated prevalence of known diabetes of 2.64%. Of these, 141 patients had insulin-dependent diabetes mellitus (IDDM) corresponding to 3.2% of identified diabetic subjects (prevalence 0.07% inhabitants); 4362 patients had non-insulin-dependent diabetes mellitus (NIDDM), 96.8% of identified diabetic subjects (prevalence 2.36%). Of patients with NIDDM 10.5% was treated by diet, 65% with oral hypoglycaemic agents (OHA), 23% with insulin and 1.5% with insulin plus OHA. This study shows that the method used in this survey is suitable for epidemiological studies because it does not demand the cooperation of the diabetic patients, is addressed to the entire diabetic population without age discrimination and singles out the diabetic population in a very reliable way.

Revisiting the association between hypnotisability and blink rate

Blink rate (BR), which is considered an index of the dopaminergic tone, has been studied in 41 subjects with high (highs), medium (mediums) and low (lows) hypnotisability scores in resting conditions. It has been found higher in highs; relaxation (indicated by skin conductance), anxiety and the proneness to absorption in tasks (assessed by questionnaires) were not responsible for the observed difference. In contrast, the BR difference did not survive controlling for mind wandering (MW questionnaire) whose variability could account for contrasting earlier reports, although no significant hypnotisability-related difference has been observed in MW scores. Findings do not allow to exclude that mechanisms other than dopaminergic ones may be involved in the observed difference in BR. In particular, we suggest that one of the mechanisms possibly sustaining the highs’ higher BR may be a reduced cerebellar inhibition. In fact, cerebellar impairment is associated with higher BR and several studies of sensorimotor integration indicate different cerebellar controls in the highs’ and lows’ behavior.

Tuning of human vestibulospinal reflexes by leg rotation

Changing the foot position modifies the mechanical action exerted by the ankle extensor and flexor muscles over the body. We verified, in two groups of healthy subjects standing with the heels touching or apart, whether a 90° external rotation of the right leg and foot also changes the pattern of vestibulospinal reflexes elicited by electrical stimulation of the labyrinth. With the head oriented forward, leg rotation did not modify the labyrinthine-driven displacements of the center of pressure (CoP). When the head was rotated in the horizontal plane, either to the right or to the left, the CoP displacement increased along the y axis in all subjects. Changes in the x component in most instances appropriate to preserve unmodified the direction of body sway elicited by the stimulus were observed. Right leg rotation increased the basal EMG activity of ankle extensors and flexors on the left side, while the right side activity was unaffected. The EMG responses to labyrinthine stimulation were modified only on the left side, in a way appropriate to correct the effects of the altered torque pattern exerted on the body by right leg muscles. It appears, therefore, that somatosensory signals related to leg rotation and/or copy of the corresponding voluntary motor commands modify the pattern of vestibulospinal reflexes and maintain the postural response appropriate to counteract a body sway in the direction inferred by labyrinthine signals.

'Gamma' band oscillatory response to chromatic stimuli in volunteers and patients with idiopathic Parkinson's disease

The signal structure of the responses to equiluminant chromatic and achromatic (contrast) stimuli was studied in normal volunteers and patients with mild to moderate idiopathic Parkinsons disease. Visual stimuli were full-field (14 x 16 deg) achromatic or equiluminant (red-green or blue-yellow) sinusoidal gratings at 2c/deg and 90% contrast presented in onset-offset mode. The signal was processed offline by DFT and factor analysis was performed in the frequency domain. The conventional VEPs to chromatic onset stimuli showed a monophasic negative wave, while the response to offset stimuli was comparable in shape to the on-/offset achromatic responses; latencies were longer and amplitudes higher than those of responses to contrast stimulation. In patients, latencies were longer than in controls after achromatic and (to a lesser extent) red-green stimulations, but not after blue-yellow stimulation; amplitudes were comparable in all stimulus conditions. In healthy subjects, two non-overlapping factors accounted for the approximately 2-30.0 Hz and approximately 25.0-50.0 Hz signal components (representative of the low-frequency VEP and gamma oscillatory responses, respectively); the frequency of the approximately 25.0-50.0 Hz factor was lower after color than after contrast stimulation. The same factor structure was identified in patients, but the peak frequency of the factor on gamma activity was higher than in controls and did not vary with color-opponent stimulation. These observations indicate that stimulus-related gamma activity originates in cortex irrespective of the activated (magno-, parvo-, or konio-cellular) visual pathway, consistent with the suggested role in the phase coding of neuronal activities. Some dopaminergic modulation of gamma activity is conceivable.